The exercise intolerance of chronic heart failure (CHF) has a substantial skeletal muscle component. Repeated contractions of skeletal muscle require precise matching of O2 delivery (QO2) - to-O2 utilization (VO2). However, CHF impairs capillary hemodynamics thereby lowering the QO2/VO2 ratio and microvascular O2 pressure and compromising blood-myocyte O2 flux. Our unique intravital microscopy model (rat spinotrapezius - a muscle amenable to exercise training) facilitates direct observation of muscle microcirculation and high temporal fidelity-determination of blood-myocyte O2 flux using phosphorescence quenching during contractions. Building on the observation that CHF decreases muscle nitric oxide (NO) bioavailability and acute increases of NO restore QO2-to-VO2 matching in CHF this proposal will test the global hypothesis that multiple strategies designed to increase NO bioavailability either directly (exercise training, increased tetrahydrobiopterin (BH4, via sepiapterin)) or via modulation of reactive O2 species ( ROS, reduced O2O-, via apocynin, Gp91 ds-tat, and Tempol;reduced OHO- via deferoxamine) will improve capillary hemodynamics and blood-myocyte O2 flux. Recognizing the inflammatory component of CHF, we will investigate whether microvascular deficits in CHF can be ameliorated by reduction of inflammatory mediators IL-12 (using anakinra, IL-12 receptor antagonist) and TNF1 (using pentoxifylline). Significant strengths of this proposal include: 1. Pentoxifylline, which lowers circulating TNF-1, has proven efficacy in human CHF whereas TNF-1 blockers etanercept (recombinant TNF receptor) and infliximab (monoclonal TNF-1 antibody), for example, may worsen the condition. We will test the hypothesis that pentoxifylline improves capillary hemodynamics during contractions and assess the role of NO in this process. 2. Involvement of undergraduate and graduate students in meritorious cutting-edge scientific research, and 3. Avoiding the technical impossibility of making precise micron-level [NO] measurements in contracting muscle by determining quantitatively the extent to which NO bioavailability actually facilitates capillary hemodynamics. This will be accomplished using conditions of NOS blockade (L-NAME) and exogenous NO application (sodium nitroprusside and/or NONOate) under each experimental condition. It is anticipated that these investigations will establish a sentinel role for NO in facilitating the capillary hemodynamics and blood-myocyte O2 flux response to muscle contractions in health and reveal that deficits in NO bioavailability in CHF - evoked by dysfunction at multiple steps in the NO production pathway including BH4-induced NOS uncoupling and ROS- mediated NO destruction - are causative to CHF-induced dysfunction. The proposed studies will provide novel and important data addressing the mechanisms of muscle capillary hemodynamic dysfunction in CHF and assess the mechanistic bases for the efficacy of pentoxifylline in treating CHF patients whilst fulfilling the AREA award mandate to integrate authentic research with undergraduate/graduate student education. PUBLIC HEALTH RELEVANCE: The exercise intolerance of patients suffering from chronic heart failure (CHF) is based substantially in skeletal muscle and, irrespective of left-ventricular function per se, results from a mismatching of oxygen delivery to oxygen demands in contracting muscles. Recent evidence indicates that this mismatching is the consequence of impaired capillary blood flow secondary to decreased nitric oxide bioavailability. This proposal assesses the efficacy of strategies to increase nitric oxide bioavailability to improve capillary hemodynamics, oxygen delivery/utilization matching and muscle function in CHF. One key aspect is determination of the mechanistic bases for the efficacy of pentoxifylline to treat CHF patients. Under these auspices and the AREA Award mission this proposal integrates meritorious science with opportunities for development of undergraduate and graduate student scientists.